An insulinotropic complex using an immunoglobulin fragment

ABSTRACT

The present invention relates to an insulinotropic peptide conjugate having improved in-vivo duration of efficacy and stability, comprising an insulinotropic peptide, a non-peptide polymer and an immunoglobulin Fc region, which are covalently linked to each other, and a use of the same. The insulinotropic peptide conjugate of the present invention has the in-vivo activity which is maintained relatively high, and has remarkably increased blood half-life, and thus it can be desirably employed in the development of long acting formulations of various peptide drugs.

This is a national stage application of PCT/KR2008/000061 filed on Jan.4, 2008, which claims priority from Korean patent application10-2007-0001662 filed Jan. 5, 2007, contents of which are incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to an insulinotropic peptide conjugate forlong acting formulation of an insulinotropic peptide. Specifically, thepresent invention relates to a modified insulinotropic peptide conjugatehaving a remarkably improved in-vivo duration of efficacy generated bycovalently linking the insulinotropic peptide with a non-peptidylpolymer and an immunoglobulin Fc, and a method for preparation thereof.

BACKGROUND ART

Peptides tend to be easily denatured due to their low stability,degraded by in-vivo proteolytic enzymes, thus losing the activity, andhave a relatively small size, thereby easily passing through the kidney.Accordingly, in order to maintain the blood levels and the titers of amedicament comprising a peptide as a pharmaceutically effectivecomponent, it is necessary to administer the peptide drug frequently toa patient to maintain desired blood levels and titers. However, thepeptide drugs are usually administered in the form of injectablepreparations, and such frequent administration cause severe pain for thepatients. To solve these problems, many efforts have been made. As oneof such efforts, there has been an attempt to transfer the peptide drugthrough oropharyngeal or nasopharyngeal inhalation by increasing thetransmission of the peptide drug through the biological membranes.However, this approach is still difficult in maintaining the in-vivoactivity of the peptide drug due to the low in-vivo transfer efficiency,as compared to the injection.

On the other hand, many efforts have been made to improve the bloodstability of the peptide drug, and to maintain the drug in the blood ata high level for a prolonged period of time, thereby maximizing thepharmaceutical efficacy of the drug. The long acting preparation of suchpeptide drug therefore needs to increase the stability of the peptidedrug, and to maintain the titers at sufficiently high levels withoutcausing immune responses in patients.

As a method for stabilizing the peptide, and inhibiting the degradationby a proteolytic enzyme, some trials have been performed to modify aspecific amino acid sequence which is sensitive to the proteolyticenzyme. For example, GLP-1 (7-37 or 7-36 amide), which functions toreduce the glucose concentration in blood for treating a Type 2diabetes, has a short half-life of the physiological activity of about 4minutes or less (Kreymann et al., 1987), due to loss of the titers ofGLP-1 through the cleavage between the 8th amino acid (Ala) and the 9thamino acid (Asp) by a dipeptidyl pepdidase IV (DPP IV). As a result,various investigations have been made on a GLP-1 analog havingresistance to DPP IV, and trials have been made for substitution of Ala⁸with Gly (Deacon et al., 1998; Burcelin et al., 1999), or with Leu orD-Ala (Xiao et al., 2001), thereby increasing the resistance to DPP IV,while maintaining the activity. The N-terminal amino acid, His⁷ of GLP-1is critical for the GLP-1 activity, and serves as a target of DPP IV.Accordingly, U.S. Pat. No. 5,545,618 describes that the N-terminus ismodified with an alkyl or acyl group, and Gallwitz, et al. describesthat 7th His was subject to N-methylation, or alpha-methylation, or theentire His is substituted with imidazole to increase the resistance toDPP IV, and to maintain physiological activity.

In addition to these modifications, an exendin-4, which is a GLP-1analog purified from the salivary gland of a glia monster (U.S. Pat. No.5,424,686), has resistance to DPP IV, and higher physiological activitythan GLP-1. As a result, it had an in-vivo half-life of 2 to 4 hours,which was longer than that of GLP-1. However, with the method forincreasing the resistance to DPP IV only, the physiological activity isnot sufficiently sustained, and for example, in the case of acommercially available exendin-4 (exenatide), it needs to be injected toa patient twice a day, which is still difficult for patients.

These insulinotropic peptides have a problem, usually in that the sizeof the peptide is small. Thus, they cannot be recovered in the kidney,and are then extracorporeally discharged. Accordingly, a method forchemically adding a polymeric substance having high solubility, such aspolyethylene glycol (PEG), onto the surface of the peptide to inhibitthe loss in the kidney, has been used.

PEG non-specifically binds to a specific site or various sites of atarget peptide to give an effect of increasing the molecular weight of apeptide, and thus inhibiting the loss by the kidney, and preventinghydrolysis, without causing any side-effects. For example, InternationalPat. Publication No. WO 2006/076471 describes that PEG binds to a B-typenatriuretic peptide, or BNP, which binds to NPR-A to activate theproduction of cGMP, which leads to reduction in the arterial bloodpressure, and as a result, is used as congestive heart failuretherapeutic agent, thereby sustaining the physiological activity. U.S.Pat. No. 6,924,264 describes that PEG binds to the lysine residue of anexendin-4 to increase its in-vivo residence time. However, this methodincreases the molecular weight of PEG, thereby increasing the in-vivoresidence time of the peptide drug, while as the molecular weight isincreased, the titer of the peptide drug is remarkably reduced, and thereactivity with the peptide is also reduced. Accordingly, it undesirablylowers the yield.

International Pat. Publication No. WO 02/46227 describes a fusionprotein prepared by coupling GLP-1, an exendin-4, or an analog thereofwith human serum albumin or an immunoglobulin region (Fc) using agenetic recombination technology. U.S. Pat. No. 6,756,480 describes anFc fusion protein prepared by coupling a parathyroid hormone (PTH) andan analog thereof with Fc region. These methods can address the problemssuch as low pegylation yield and non-specificity, but they still have aproblem in that the effect of increasing the blood half-life is notnoticeable as expected, and sometimes the titers are also low. In orderto maximize the effect of increasing the blood half-life, various kindsof peptide linkers are used, but an immune response may be possiblycaused. Further, if a peptide having disulfide bonds, such as BNP isused, there is a high probability of misfolding. As a result, suchpeptide can hardly be used.

In addition, a GLP-1 derivative, NN2211, is prepared by substitution ofthe amino acid of GLP-1, and is bound to an acyl side chain to form anon-covalent bond with albumin, thereby increasing its in-vivo residencetime. However, it has a half-life of 11 to 15 hours, which does notindicate remarkable increase in the half-lives, as compared with theexendin-4. Thus, the GLP-1 derivative still needs to be injected once aday (Nauck et al., 2004). Further, CJC-1131 is a GLP-1 derivative havinga maleimide reactive group for covalently binding the GLP-1 with albuminin blood, and efforts had been tried to develop the CJC-1131 for thepurpose of increasing the in-vivo half-life, but such efforts were nowstopped. A subsequently suggested substance, CJC-1134, is an exendin-4which covalently binds to a recombinant albumin, and did not exhibit aremarkable effect of increasing blood stability, with the bloodhalf-life being about 17 hours (Rat) (Thibauoleau et. al., 2006).

DISCLOSURE Technical Problem

Thus, the present inventors linked an immunoglobulin Fc, and anon-peptidyl polymer, to an insulinotropic peptide site-specifically atan amino acid residue other than the N-terminus by a covalent bond, andfound that the conjugate of the present invention exerts a remarkablyincreased in-vivo efficacy and half life. Especially, they have foundthat, among the insulinotropic peptide conjugates, the conjugates of thepeptides such as Exendin-4, des-amino-histidyl exendin-4 where theN-terminal amine group of exendin-4 is deleted,beta-hydroxy-imidazo-propionyl exendin-4 where the N-terminal aminegroup of exendin-4 is substituted with hydroxyl group, dimethyl-histidylexendin-4 where the N-terminal amine group of exendin-4 is modified withtwo methyl groups, and an imidazo-acetyl-exendin-4 where the alphacarbon of the first histidine and the N-terminal amine group likedthereto are deleted, exert a remarkably increased in-vivo efficacy andhalf life.

Technical Solution

It is an object of the present invention to provide a long actingpreparation of insulinotropic peptide, having the effects of maintainingthe in-vivo activity of the insulinotropic peptide and increasing theblood half-life.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of reverse phase HPLC for measurement of thepurity of a native exendin-4(N)-PEG-immunoglobulin Fc conjugate;

FIG. 2 shows the results of reverse phase HPLC for measurement of thepurity of a native exendin-4(Lys)-PEG-immunoglobulin Fc conjugate;

FIG. 3 shows the results of reverse phase HPLC for measurement of thepurity of a des-amino-histidyl exendin-4(Lys)-PEG-immunoglobulin Fcconjugate;

FIG. 4 shows the results of reverse phase HPLC for measurement of thepurity of a(2-Hydroxy-3-(1H-imidazol-4-yl)propionyl)exendin-4(Lys)-PEG-immunoglobulinFc conjugate;

FIG. 5 shows the results of reverse phase HPLC for measurement of thepurity of a(2-(1H-imidazol-4-yl)acetyl)exendin-4(Lys)-PEG-immunoglobulin Fcconjugate;

FIG. 6 shows the results of reverse phase HPLC for measurement of thepurity of a Ser12 mutated des-amino-histidylexendin-4(Lys)-PEG-immunoglobulin Fc conjugate;

FIG. 7 shows the results of reverse phase HPLC for measurement of thepurity of an Arg12-mutated des-amino-histidylexendin-4(Lys)-PEG-immunoglobulin Fc conjugate;

FIG. 8 shows the results of reverse phase HPLC for measurement of thepurity of a des-amino-histidyl exendin-4(Lys)-PEG-human serum albumin(HSA) conjugate;

FIG. 9 shows the results of reverse phase HPLC for measurement of thepurity of a dimethyl-histidyl exendin-4(Lys)-PEG-immunoglobulin Fcconjugate;

FIG. 10 shows the results of reverse phase HPLC for measurement of thepurity of a GLP-1(N)-PEG-immunoglobulin Fc conjugate;

FIG. 11 shows the results of reverse phase HPLC for measurement of thepurity of a des-amino-histidyl GLP-1(Lys)-PEG-immunoglobulin Fcconjugate;

FIG. 12 shows the results of reverse phase HPLC for measurement of thepurity of a native exendin-4(Lys)-PEG-immunoglobulin Fc conjugate;

FIG. 13 shows the results of measurement of the purity of a(2-(1H-imidazol-4-yl)acetyl)exendin-4(Lys)-PEG-immunoglobulin Fcconjugate by 12% SDS-PAGE; and

FIG. 14 shows the results of measurement of the glucose concentrationreducing effect in blood of a des-amino-histidylexendin-4(Lys)-PEG-immunoglobulin Fc conjugate. In FIG. 14, HM11260A isexendin-4 (Lys27)-PET-Fc; HM11260D is DA exendin-4 (Lys27)-PET-Fc;HM11260DM is DM exendin-4 (Lys27)-PET-Fc; HM11260S is Ser12 DA exendin-4(Lys27)-PET-Fc; HM 11260C is CA exendin-4 (Lys27)-PET-Fc; and HM11260His HY exendin-4 (Lys27)-PET-Fc.

DETAILED DESCRIPTION

In one embodiment of the present invention, there is provided a longacting insulinotropic peptide conjugate, in which an insulinotropicpeptide and a non-peptidyl polymer possessing a reactive group at bothends thereof are covalently linked to each other.

The insulinotropic peptide of the present invention is a peptidepossessing an insulinotropic function for promoting the synthesis andthe expression of insulin in a pancreatic beta cell. These peptidesinclude a precursor, an agonist, a derivative, a fragment, and avariant, and preferably GLP (glucagon like peptide)-1, exendin 3, andexendin 4.

GLP-1 is a hormone that is secreted by the small intestine, generallypromotes the biosynthesis and secretion of insulin, inhibits thesecretion of glucagon, and promotes glucose absorption in the cells. Inthe small intestine, a glucagon precursor is decomposed into threepeptides, that is, glucagon, GLP-1, and GLP-2. Here, the GLP-1 meansGLP-1 (1-37), which is originally in the form having no insulinotropicfunction. But it is then processed and converted into one in theactivated GLP-1 (7-37) form. The sequence of the GLP-1 (7-37) amino acidis as follows:

GLP-1(7-37)

(SEQ ID NO: 1) HAEGT FTSDV SSYLE GQAAK EFIAW LVKGR G

The GLP-1 derivative means a peptide which exhibits an amino acidsequence homology of at least 80% with that of GLP-1, may be in thechemically modified form, and exhibits an insulinotropic function of atleast equivalent or more to that of GLP-1.

The GLP-1 fragment means one in the form in which one or more aminoacids are added or deleted at an N-terminus or a C-terminus of a nativeGLP-1, wherein the added amino acid is possibly non-naturally occurringamino acid (e.g., D-type amino acid). The GLP-1 variant means a peptidepossessing an insulinotropic function, which has one or more amino acidsequences different from those of a native GLP-1.

The exendin 3 and the exendin 4 are insulinotropic peptides consistingof 39 amino acids, which have a 53% amino acid sequence homology withGLP-1. The amino acid sequences of the exendin-3 and the exendin-4 areas follows:

Exendin-3 (SEQ ID NO: 2) HSDGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPSExendin-4 (SEQ ID NO: 3) HGEGT FTSDL SKQME EEAVR LFIEW LKNGG PSSGA PPPS

The exendin agonist means a compound reacting with receptors in-vivo andhaving equal biological activity to that of exendin, which is irrelevantto the structure of exendin. The exendin derivative means a peptidehaving at least 80% amino acid sequence homology with the nativeexendin, which may have some groups on the amino acid residue chemicallysubstituted (e.g., alpha-methylation, alpha-hydroxylation), deleted(e.g., deamination), or modified (e.g., N-methylation), and has aninsulinotropic function.

The exendin fragment means a fragment having one or more amino acidsadded or deleted at the N-terminus or the C-terminus of the nativeexendin, in which non-naturally occurring amino acids (for example,D-type amino acid) can be added, and has an insulinotropic function.

The exendin variant means a peptide having at least one amino acidsequence different from that of the native exendin, in which has aninsulinotropic function. Each of the preparation methods for the exendinagonist, derivative, the fragment, and the variant can be usedindividually or in combination. For example, the present inventionincludes an insulinotropic peptide having an amino acid sequence whichhave at least one different amino acids from those of nativeinsulinotropic peptide, and having the amino acid residue at theN-terminus deaminated.

In a specific embodiment, the native insulinotropic peptide used in thepresent invention, and the modified insulinotropic peptide can besynthesized using a solid phase synthesis method, and most of the nativepeptides including a native insulinotropic peptide can be produced by arecombination technology.

Further, the insulinotropic peptide used in the present invention canbind to the non-peptidyl polymer on various sites.

The peptide conjugate prepared according to the present invention canhave an activity which varies depending on the sites to be linked to theinsulinotropic peptide. For example, it can be coupled with anN-terminus, and other terminus other than the N-terminus, such as aC-terminus, respectively, which indicates difference in the in vitroactivity. The aldehyde reactive group selectively binds to an N-terminusat a low pH, and can bind to a lysine residue to form a covalent bond ata high pH, such as pH 9.0. A pegylation reaction is allowed to proceedwith varying pH, and then a positional isomer can be separated from thereaction mixture using an ion exchange column.

If the insulinotropic peptide is to be coupled at a site other than theN-terminus which is an important site for the in-vivo activity, areactive thiol group can be introduced to the site of amino acid residueto be modified in the native amino acid sequence to form a covalent bondusing a maleimide linker at the non-peptidyl polymer.

If the insulinotropic peptide is to be coupled at a site other than theN-terminus which is an important site for the in-vivo activity, areactive amine group can be introduced to the site of amino acid residueto be modified in the native amino acid sequence to form a covalent bondusing an aldehyde linker at the non-peptidyl polymer.

When the aldehyde linker at the non-peptidyl polymer is used, it isreacted with an amino group at the N-terminus and the lysine residue,and a modified form of the insulinotropic peptide can be used toselectively increase the reaction yield. For example, only one aminegroup to be reacted can be retained on a desired site, using anN-terminus blocking method, a lysine residue substituting method, amethod for introducing an amine group at a carboxyl terminus, or thelike, thereby increasing the yield of pegylation and coupling reactions.The methods for protecting the N-terminus include dimethylation, as wellas methylation, deamination, acetylation, etc., but are not limited tosuch alkylation methods.

In one preferable embodiment, the insulinotropic peptide conjugate ofthe present invention is an insulinotropic peptide conjugate, in whichan immunoglobulin Fc region specifically binds to an amine group otherthan ones at the N-terminus of the insulinotropic peptide.

In one specific embodiment, the present inventors induced a pegylationof a native exendin-4 at pH 9.0, to selectively couple the PEG to thelysine residue of the insulinotropic peptide. Alternatively, to pegylateat Lys residue, the pegylation of the exendin-4 derivatives having theN-terminus deleted or protected was performed at pH 7.5. The pegylationat the N-terminus was blocked, either by deleting the alpha amine groupof the N-terminal histidine, by substituting the N-terminal amine groupwith hydroxyl group, by modifying the alpha amine group of N-terminalhistidine with two methyl groups, or by deleting the alpha carbon of thefirst amino acid (histidine) and the N-terminal amine group linkedthereto to leave the imidazo-acetyl group, and etc. Such derivativeswere represented by the following Chemical Formulas:

Unlike the N-terminal coupling, when the PEG was coupled to the lysineresidue rather than the N-terminus, the in vitro activity was maintainedat about 8.5% (Table 1). Further, even if the Fc conjugates ofdes-amino-histidyl exendin-4 (hereinafter, referred to as DA-exendin-4)prepared by deleting N-terminal amine group of exendin-4,beta-hydroxyl-imidazol propionyl exendin-4 (hereinafter, referred to asHY-exendin-4) prepared by substituting N-terminal amine group ofexendin-4 with hydroxyl group, dimethyl histidyl exendin-4 (hereinafter,referred to as DM-exendin-4) prepared by modifying N-terminal aminegroup of exendin-4 with two methyl group, and imidazolacetyl-exendin-4(hereinafter, referred to as CA-exendin-4) prepared by deleting α-carbonof the first histidine of exendin-4 and the N-terminal amine grouplinked thereto, showed comparable in-vitro activity and the blood halflife to the native exendin-4 conjugate (Table 1), these conjugatesshowed unexpectedly high in-vivo duration of efficacy (FIG. 14).DM-exendin-4-immunoglobulin Fc conjugate, DA-exendin-4-immunoglobulin Fcconjugate, CA-exendin-4-immunoglobulin Fc conjugate and HYexendin-4-immunoglobulin Fc conjugate prepared according to the presentinvention showed an increased blood half-life of 50 hours or more. Thetiter reduction was also minimized by coupling to the Lys residue whichdoes not affect the activity of the peptide. In addition, anunexpectedly high glucose lowering activity was observed by removing theamine group or alpha carbon at the N-terminus.

The immunoglobulin Fc region is safe for use as a drug carrier becauseit is a biodegradable polypeptide that is in vivo metabolized. Also, theimmunoglobulin Fc region has a relatively low molecular weight, ascompared to the whole immunoglobulin molecules, and thus, it isadvantageous in the preparation, purification and yield of theconjugate. Since the immunoglobulin Fc region does not contain a Fabfragment, whose amino acid sequence differs according to the antibodysubclasses and which thus is highly non-homogenous, it can be expectedthat the immunoglobulin Fc region may greatly increase the homogeneityof substances and be less antigenic.

The insulinotropic peptide used in the present invention is linked witha carrier substance and non-peptidyl polymer.

The carrier substance which can be used in the present invention may beselected from the group consisting of an immunoglobulin Fc region, analbumin, a transferrin, and a PEG, and preferably an immunoglobulin Fcregion.

The term “immunoglobulin Fc region” as used herein, refers to theheavy-chain constant region 2 (C_(H)2) and the heavy-chain constantregion 3 (C_(H)3) of an immunoglobulin, and not the variable regions ofthe heavy and light chains, the heavy-chain constant region 1 (C_(H)1)and the light-chain constant region 1 (C_(L)1) of the immunoglobulin. Itmay further include a hinge region at the heavy-chain constant region.Also, the immunoglobulin Fc region of the present invention may containa part or all of the Fc region including the heavy-chain constant region1 (C_(H)1) and/or the light-chain constant region 1 (C_(L)1), except forthe variable regions of the heavy and light chains, as long as it has aphysiological function substantially similar to or better than thenative protein. Also, the Ig Fc region may be a fragment having adeletion in a relatively long portion of the amino acid sequence ofC_(H)2 and/or C_(H)3. That is, the immunoglobulin Fc region of thepresent invention may comprise 1) a C_(H)1 domain, a C_(H)2 domain, aC_(H)3 domain and a C_(H)4 domain, 2) a C_(H)1 domain and a C_(H)2domain, 3) a C_(H)1 domain and a C_(H)3 domain, 4) a C_(H)2 domain and aC_(H)3 domain, 5) a combination of one or more domains and animmunoglobulin hinge region (or a portion of the hinge region), and 6) adimer of each domain of the heavy-chain constant regions and thelight-chain constant region.

The immunoglobulin Fc region of the present invention includes a nativeamino acid sequence, and a sequence derivative (mutant) thereof. Anamino acid sequence derivative is a sequence that is different from thenative amino acid sequence due to a deletion, an insertion, anon-conservative or conservative substitution or combinations thereof ofone or more amino acid residues. For example, in an IgG Fc, amino acidresidues known to be important in binding, at positions 214 to 238, 297to 299, 318 to 322, or 327 to 331, may be used as a suitable target formodification. Also, other various derivatives are possible, includingone in which a region capable of forming a disulfide bond is deleted, orcertain amino acid residues are eliminated at the N-terminus of a nativeFc form or a methionine residue is added thereto. Further, to removeeffector functions, a deletion may occur in a complement-binding site,such as a C1q-binding site and an ADCC (antibody dependent cell mediatedcytotoxicity) site. Techniques of preparing such sequence derivatives ofthe immunoglobulin Fc region are disclosed in International Pat.Publication Nos. WO 97/34631 and WO 96/32478.

Amino acid exchanges in proteins and peptides, which do not generallyalter the activity of molecules are known in the art (H. Neurath, R. L.Hill, The Proteins, Academic Press, New York, 1979). The most commonlyoccurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly in both directions.

The Fc region, if desired, may be modified by phosphorylation,sulfation, acrylation, glycosylation, methylation, farnesylation,acetylation, amidation, and the like.

The aforementioned Fc derivatives are derivatives that have a biologicalactivity identical to the Fc region of the present invention or improvedstructural stability, for example, against heat, pH, or the like.

In addition, these Fc regions may be obtained from native forms isolatedfrom humans and other animals including cows, goats, swine, mice,rabbits, hamsters, rats and guinea pigs, or may be recombinants orderivatives thereof, obtained from transformed animal cells ormicroorganisms. Herein, they may be obtained from a nativeimmunoglobulin by isolating whole immunoglobulins from human or animalorganisms and treating them with a proteolytic enzyme. Papain digeststhe native immunoglobulin into Fab and Fc regions, and pepsin treatmentresults in the production of pF′c and F(ab)₂ fragments. These fragmentsmay be subjected, for example, to size exclusion chromatography toisolate Fc or pF′c.

Preferably, a human-derived Fc region is a recombinant immunoglobulin Fcregion that is obtained from a microorganism.

In addition, the immunoglobulin Fc region of the present invention maybe in the form of having native sugar chains, increased sugar chainscompared to a native form or decreased sugar chains compared to thenative form, or may be in a deglycosylated form. The increase, decreaseor removal of the immunoglobulin Fc sugar chains may be achieved bymethods common in the art, such as a chemical method, an enzymaticmethod and a genetic engineering method using a microorganism. Theremoval of sugar chains from an Fc region results in a sharp decrease inbinding affinity to the C1q part of the first complement component C1and a decrease or loss in antibody-dependent cell-mediated cytotoxicityor complement-dependent cytotoxicity, thereby not inducing unnecessaryimmune responses in-vivo. In this regard, an immunoglobulin Fc region ina deglycosylated or aglycosylated form may be more suitable to theobject of the present invention as a drug carrier.

As used herein, the term “deglycosylation” refers to enzymaticallyremove sugar moieties from an Fc region, and the term “aglycosylation”means that an Fc region is produced in an unglycosylated form by aprokaryote, preferably E. coli.

On the other hand, the immunoglobulin Fc region may be derived fromhumans or other animals including cows, goats, swine, mice, rabbits,hamsters, rats and guinea pigs, and preferably humans. In addition, theimmunoglobulin Fc region may be an Fc region that is derived from IgG,IgA, IgD, IgE and IgM, or that is made by combinations thereof orhybrids thereof. Preferably, it is derived from IgG or IgM, which isamong the most abundant proteins in human blood, and most preferablyfrom IgG, which is known to enhance the half-lives of ligand-bindingproteins.

On the other hand, the term “combination” as used herein, means thatpolypeptides encoding single-chain immunoglobulin Fc regions of the sameorigin are linked to a single-chain polypeptide of a different origin toform a dimer or multimer. That is, a dimer or multimer may be formedfrom two or more fragments selected from the group consisting of IgG Fc,IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

The term “hybrid” as used herein, means that sequences encoding two ormore immunoglobulin Fc regions of different origin are present in asingle-chain immunoglobulin Fc region. In the present invention, varioustypes of hybrids are possible. That is, domain hybrids may be composedof one to four domains selected from the group consisting of C_(H)1,C_(H)2, C_(H)3 and C_(H)4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc,and may include the hinge region.

On the other hand, IgG is divided into IgG1, IgG2, IgG3 and IgG4subclasses, and the present invention includes combinations and hybridsthereof. Preferred are IgG2 and IgG4 subclasses, and most preferred isthe Fc region of IgG4 rarely having effector functions such as CDC(complement dependent cytotoxicity).

That is, as the drug carrier of the present invention, the mostpreferable immunoglobulin Fc region is a human IgG4-derivednon-glycosylated Fc region. The human-derived Fc region is morepreferable than a non-human derived Fc region, which may act as anantigen in the human body and cause undesirable immune responses such asthe production of a new antibody against the antigen.

The term “non-peptidyl polymer” as used herein, refers to abiocompatible polymer including two or more repeating units linked toeach other by any covalent bond excluding a peptide bond.

The non-peptidyl polymer which can be used in the present invention maybe selected form the group consisting of polyethylene glycol,polypropylene glycol, copolymers of ethylene glycol and propyleneglycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides,dextran, polyvinyl ethyl ether, biodegradable polymers such as PLA(polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers,chitins, hyaluronic acid, and combinations thereof, and preferred ispolyethylene glycol. Also, derivatives thereof well known in the art andbeing easily prepared within the skill of the art are included in thescope of the present invention.

The peptide linker which is used in the fusion protein obtained by aconventional inframe fusion method has drawbacks that it is easilyin-vivo cleaved by a proteolytic enzyme, and thus a sufficient effect ofincreasing the blood half-life of the active drug by a carrier cannot beobtained as expected. However, in the present invention, a polymerhaving resistance to the proteolytic enzyme can be used to maintain theblood half-life of the peptide to be similar to that of the carrier.Therefore, any non-peptidyl polymer which can be used in the presentinvention can be used without any limitation, as long as it is a polymerhaving the aforementioned function, that is, a polymer having resistanceto the in-vivo proteolytic enzyme. The non-peptidyl polymer preferablyhas a molecular weight in the range of 1 to 100 kDa, and preferably of 1to 20 kDa. Also, the non-peptidyl polymer of the present invention,linked to the immunoglobulin Fc region, may be one polymer or acombination of different types of polymers.

The non-peptidyl polymer used in the present invention has a reactivegroup capable of binding to the immunoglobulin Fc region and the proteindrug.

The non-peptidyl polymer has a reactive group at both ends, which ispreferably selected from the group consisting of a reactive aldehydegroup, a propionaldehyde group, a butyraldehyde group, a maleimide groupand a succinimide derivative. The succinimide derivative may besuccinimidyl propionate, hydroxy succinimidyl, succinimidylcarboxymethyl, or succinimidyl carbonate. In particular, when thenon-peptidyl polymer has a reactive aldehyde group at both ends, it iseffective in linking at both ends with a physiologically activepolypeptide and an immunoglobulin with minimal non-specific reactions. Afinal product generated by reductive alkylation by an aldehyde bond ismuch more stable than when linked by an amide bond. The aldehydereactive group selectively binds to an N-terminus at a low pH, and canbind to a lysine residue to form a covalent bond at a high pH, such aspH 9.0.

The reactive groups at both ends of the non-peptidyl polymer may be thesame or different. For example, the non-peptide polymer may possess amaleimide group at one end and at the other end, an aldehyde group, apropionaldehyde group or a butyraldehyde group. When a polyethyleneglycol having a reactive hydroxy group at both ends thereof is used asthe non-peptidyl polymer, the hydroxy group may be activated to variousreactive groups by known chemical reactions, or a polyethylene glycolhaving a commercially available modified reactive group may be used soas to prepare the insulinotropic peptide conjugate of the presentinvention.

The insulinotropic peptide conjugate of the present invention maintainsthe conventional in-vivo activities of the insulinotropic peptide, suchas promotion of synthesis and secretion of insulin, appetite control,weight loss, increase in the beta cell sensitivity to glucose in blood,promotion of beta cell proliferation, delayed gastric emptying, andglucagon suppression, and further remarkably increases the bloodhalf-life of the insulinotropic peptide, and hence the in-vivo efficacysustaining effect of the peptide, it is useful to treat diabetes,obesity, acute coronary syndrome, or polycystic ovary syndrome.

In another embodiment, the present invention provides a method forpreparing an insulinotropic peptide conjugate, comprising the steps of:

(1) covalently linking a non-peptidyl polymer having a reactive groupselected from the group consisting of aldehyde, maleimide, andsuccinimide derivatives at both ends thereof, with an amine group orthiol group of the insulinotropic peptide;(2) isolating a conjugate comprising the insulinotropic peptide from thereaction mixture of (1), in which the non-peptidyl polymer is linkedcovalently to a site other than the N-terminus; and(3) covalently linking an immunoglobulin Fc region to the other end ofthe non-peptidyl polymer of the isolated conjugate to produce a peptideconjugate comprising the immunoglobulin Fc region and the insulinotropicpeptide, which are linked to each end of the non-peptidyl polymer.

The term “conjugate” as used herein, refers to an intermediate preparedby covalently linking the non-peptidyl polymer with the insulinotropicpeptide, and subsequently the immunoglobulin Fc region is linked to theother end of the non-peptidyl polymer.

In a preferable embodiment, the present invention provides a method forpreparing an insulinotropic peptide conjugate, comprising the steps of:

(1) covalently linking a non-peptidyl polymer having an aldehydereactive group at both ends thereof with the lysine residue of theinsulinotropic peptide;(2) isolating a conjugate comprising the insulinotropic peptide from thereaction mixture of (1), in which the non-peptidyl polymer is linkedcovalently to the lysine residue; and(3) covalently linking an immunoglobulin Fc region to the other end ofthe non-peptidyl polymer of the isolated conjugate to produce a proteinconjugate comprising the immunoglobulin Fc region and the insulinotropicpeptide, which are linked to each end of the non-peptidyl polymer. Morepreferably, the non-peptidyl polymer of (1), and the lysine residue ofthe insulinotropic peptide are linked at pH 7.5 or higher.

In a further embodiment, the present invention provides a pharmaceuticalcomposition for treating diabetes, comprising the insulinotropic peptideconjugate of the present invention.

The pharmaceutical composition comprising the conjugate of the presentinvention can further comprise a pharmaceutically acceptable carrier.For oral administration, the pharmaceutically acceptable carrier mayinclude a binder, a lubricant, a disintegrator, an excipient, asolubilizer, a dispersing agent, a stabilizer, a suspending agent, acoloring agent, and a perfume. For injectable preparations, thepharmaceutically acceptable carrier may include a buffering agent, apreserving agent, an analgesic, a solubilizer, an isotonic agent, and astabilizer. For preparations for topical administration, thepharmaceutically acceptable carrier may include a base, an excipient, alubricant, and a preserving agent. The pharmaceutical composition of thepresent invention may be formulated into a variety of dosage forms incombination with the aforementioned pharmaceutically acceptablecarriers. For example, for oral administration, the pharmaceuticalcomposition may be formulated into tablets, troches, capsules, elixirs,suspensions, syrups or wafers. For injectable preparations, thepharmaceutical composition may be formulated into an ampule as asingle-dose dosage form or a unit dosage form, such as a multidosecontainer. The pharmaceutical composition may be also formulated intosolutions, suspensions, tablets, pills, capsules and long-actingpreparations.

On the other hand, examples of the carrier, the excipient, and thediluent suitable for the pharmaceutical formulations include lactose,dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol,starch, acacia rubber, alginate, gelatin, calcium phosphate, calciumsilicate, cellulose, methylcellulose, microcrystalline cellulose,polyvinylpyrrolidone, water, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate and mineral oils. Inaddition, the pharmaceutical formulations may further include fillers,anti-coagulating agents, lubricants, humectants, perfumes, andantiseptics.

The conjugate according to the present invention is useful to treatdiabetes, obesity, acute coronary syndrome, or polycystic ovarysyndrome. Accordingly, a pharmaceutical composition comprising theconjugate can be administered for the treatment of the diseases.

The term “administration” as used herein, means introduction of apredetermined amount of a substance into a patient by a certain suitablemethod. The conjugate of the present invention may be administered viaany of the common routes, as long as it is able to reach a desiredtissue. A variety of modes of administration are contemplated, includingintraperitoneally, intravenously, intramuscularly, subcutaneously,intradermally, orally, topically, intranasally, intrapulmonarily andintrarectally, but the present invention is not limited to theseexemplified modes of administration. However, since peptides aredigested upon oral administration, active ingredients of a compositionfor oral administration should be coated or formulated for protectionagainst degradation in the stomach. Preferably, the present compositionmay be administered in an injectable form. In addition, thepharmaceutical composition of the present invention may be administeredusing a certain apparatus capable of transporting the active ingredientsinto a target cell.

The administration frequency and dose of the pharmaceutical compositionof the present invention can be determined by several related factorsincluding the types of diseases to be treated, administration routes,the patient's age, gender, weight and severity of the illness, as wellas by the types of the drug as an active component. Since thepharmaceutical composition of the present invention has excellentduration of in-vivo efficacy and titer, it can remarkably reduce theadministration frequency and dose of pharmaceutical drugs of the presentinvention.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

MODE FOR INVENTION Example 1 Pegylation of exendin-4 and isolation ofpositional isomer

3.4K PropionALD(2) PEG (PEG having two propionaldehyde groups, IDB Inc.,South Korea) and the N-terminus of the exendin-4 (AP, USA) weresubjected to pegylation by reacting the peptide and the PEG at 4° C. for90 min at a molar ratio of 1:15, with a peptide concentration of 3mg/ml. At this time, the reaction was performed in a NaOAc buffer at pH4.0 at a concentration of 100 mM, and 20 mM SCB (NaCNBH3) as a reducingagent was added thereto to perform the reaction. 3.4K PropionALD(2) PEGand the lysine (Lys) residue of the exendin-4 were subjected topegylation by reacting the peptide and the PEG at 4° C. for 3 hours at amolar ratio of 1:30, with a peptide concentration of 3 mg/ml. At thistime, the reaction was performed in a Na-Phosphate buffer at pH 9.0 at aconcentration of 100 mM, and 20 mM SCB as a reducing agent was addedthereto to perform the reaction. A mono-pegylated peptide was purifiedfrom each of the reaction solutions using SOURCE Q (XK 16 ml, AmershamBiosciences), and isomers were isolated using SOURCE S (XK 16 ml,Amersham Biosciences). It was found that a peak for pegylated N-terminuswas found earlier, and then two peaks for pegylated lysine residues werefound in turn. The pegylated regions of eluted peaks were confirmed bypeptide mapping method. The Lys12-pegylated conjugate was elutedearlier, the Lys 27-pegylated conjugate was eluted in the last portion,and a positional isomer of N-terminus and a positional isomer of Lys 12are completely isolated from each other.

Column: SOURCE Q (XK 16 ml, Amersham Biosciences)

Flow rate: 2.0 ml/min

Gradient: A 0->40% 80 min B (A: 20 mM Tris pH 8.5, B: A+0.5 M NaCl)Column: SOURCE S (XK 16 ml, Amersham Biosciences)

Flow rate: 2.0 ml/minGradient: A 0->100% 50 min B (A: 20 mM citric acid pH 3.0, B: A+0.5 MKCl)

Example 2 Preparation of exendin-4(N)-PEG-immunoglobulin Fc conjugate

Using the same method as described in EXAMPLE 1, 3.4K PropionALD(2) PEGand the N-terminus of the exendin-4 were reacted, and only theN-terminal isomers were purified, and then coupled with immunoglobulinFc. The reaction was performed at a ratio of peptide:immunoglobulin Fcof 1:8, and a total concentration of proteins of 50 mg/ml at 4° C. for17 hours. The reaction was performed in a solution of 100 mM K—P (pH6.0), and 20 mM SCB as a reducing agent was added thereto. The couplingreaction solution was purified using two purification columns. First,SOURCE Q (XK 16 ml, Amersham Biosciences) was used to remove a largeamount of immunoglobulin Fc which had not participated in the couplingreaction. Using 20 mM Tris (pH 7.5) and 1 M NaCl with salt gradients,the immunoglobulin Fc having relatively weak binding power was elutedearlier, and then the exendin-4-immunoglobulin Fc was eluted. Throughthis first purification procedure, the immunoglobulin Fc was removed tosome degree, but since the immunoglobulin Fc and theexendin-4-immunoglobulin Fc have similar binding powers to each other inthe ion exchange column, they could not be completely separated fromeach other. Accordingly, secondary purification was performed usinghydrophobicity of each of two materials. Using 20 mM Tris (pH7.5) 1.5 Mammonium sulfate in SOURCE ISO(HR 16 ml, Amersham Biosciences), thefirst purified samples were coupled, and the sample was eluted withgradually reducing the concentration of ammonium sulfate. In the HICColumn, the immunoglobulin Fc having weak binding power was elutedearlier, and then the exendin-4-immunoglobulin Fc sample having strongbinding power was eluted. Since they have prominently differenthydrophobicity, they can be more easily separated from each other thanin the ion exchange column. However, because excess amounts ofimmunoglobulin Fc is used due to the difference of molar ratio, highpurity was not obtainable using only HIC column. The purity measured byreverse phase HPLC was 91.6%. (FIG. 1)

Column: SOURCE Q (XK 16 ml, Amersham Biosciences)

Flow rate: 2.0 ml/min

Gradient: A 0->25% 70 min B (A: 20 mM Tris pH7.5, B: A+1 M NaCl) Column:SOURCE ISO(HR 16 ml, Amersham Biosciences)

Flow rate: 7.0 ml/minGradient: B 100->0% 60 min B (A: 20 mM Tris pH7.5, B: A+1.5 M ammoniumsulfate)

Example 3 Preparation of exendin-4(Lys27)-PEG-immunoglobulin Fcconjugate

Using the same method as described in EXAMPLE 1, 3.4K PropionALD(2) PEGand the lysine (Lys) of the exendin-4 were reacted, and only the Lysisomers were purified, and then coupled with immunoglobulin Fc. Amongthe two isomer peaks, the last isomer peak (positional isomer of Lys27),which has more reaction and which is easily distinguishable from theN-terminal isomer peaks, was used for the coupling reaction. Thereaction was performed at a ratio of peptide:immunoglobulin Fc of 1:8,and a total concentration of proteins of 50 mg/ml at 4° C. for 16 hours.The reaction was performed in a solution of 100 mM K—P (pH 6.0), and 20mM SCB was added as a reducing agent. After the coupling reaction, thetwo-step purification process using SOURCE Q 16 ml and SOURCE ISO 16 mlwas the same as in EXAMPLE 2. The purity measured by reverse phase HPLCwas 91.7%. (FIG. 2)

Example 4 Preparation of des-amino-histidylexendin-4(Lys27)-PEG-immunoglobulin Fc conjugate

To pegylate 3.4K PropionALD(2) PEG to the lysine (Lys) residue of thedes-amino-histidyl exendin-4 (DA-exedin-4, AP, USA), pegylation wasperformed with by reacting the peptide and the 3.4K PropionALD(2) at 4°C. for 12 hours at a molar ratio of 1:30, with a peptide concentrationof 3 mg/ml. The reaction solution was in a Na-Phosphaste buffer, pH 7.5at 100 mM, and 20 mM SCB was added as a reducing agent. A pegylatedpeptide was purified by the two-step purification process, using SOURCEQ (XK 16 ml, Amersham Biosciences) and SOURCE S (XK 16 ml, AmershamBiosciences). Among the two isomer peaks, the last isomer peak(positional isomer of Lys27), which has more reaction and which iseasily distinguishable from the N-terminal isomer peaks, was used forthe coupling reaction. The reaction was performed at a ratio ofpeptide:immunoglobulin Fc of 1:8, at a total proteins concentration of60 mg/ml, at 4° C. for 20 hours. The reaction was performed in 100 mMK—P (pH 6.0), and 20 mM SCB was added as a reducing agent. The two-steppurification process using SOURCE Q 16 ml and SOURCE ISO 16 ml, afterthe coupling, was the same as described in EXAMPLE 2. The puritymeasured by reverse phase HPLC was 95.8%. (FIG. 3)

Column: SOURCE Q (XK 16 ml, Amersham Biosciences)

Flow rate: 2.0 ml/min

Gradient: A 0->20% 70 min B (A: 20 mM Tris pH9.0, B: A+1 M NaCl) Column:SOURCE S (XK 16 ml, Amersham Biosciences)

Flow rate: 2.0 ml/minGradient: A 0->50% 50 min B (A: 20 mM Citric acid pH3.0, B: A+1 M KCl)

Example 5 Preparation of hydroxyl-imidazo-propionylexendin-4(Lys27)-PEG-immunoglobulin Fc conjugate

Using the same method as described in EXAMPLE 4, 3.4K PropionALD(2) PEGwas reacted with the lysine (Lys) residue of thebeta-hydroxy-imidazo-propionyl exendin-4(HY-exedin-4, AP, USA). Amongthe two isomer peaks, the last isomer peak (positional isomer of Lys27),which has more reaction and which is easily distinguishable from theN-terminal isomer peaks, was used for the coupling reaction. Thereaction was performed at a ratio of peptide:immunoglobulin Fc of 1:8,and a total protein concentration of 60 mg/ml, at 4° C. for 20 hours.The reaction was performed in 100 mM K—P (pH 6.0), and 20 mM SCB wasadded as a reducing agent. After the coupling reaction, the two-steppurification was performed using SOURCE Q 16 ml and SOURCE ISO 16 ml, asdescribed in EXAMPLE 2. The purity measured by reverse phase HPLC was93.9%. (FIG. 4)

Example 6 Preparation of imidazo-acetylexendin-4(Lys27)-PEG-immunoglobulin Fc conjugate

Using the same method as described in EXAMPLE 4, 3.4K PropionALD(2) PEGwas reacted with the lysine (Lys) residue of the imidazo-acetylexendin-4(CA-exedin-4, AP, USA). Among the two isomer peaks, the lastisomer peak (positional isomer of Lys27), which has more reaction andwhich is easily distinguishable from the N-terminal isomer peaks, wasused for the coupling reaction. The reaction was performed at a ratio ofpeptide:immunoglobulin Fc of 1:8, and a total protein concentration of60 mg/ml, at 4° C. for 20 hours. The reaction was performed in 100 mMK—P (pH 6.0), and 20 mM SCB was added as a reducing agent. After thecoupling reaction, the two-step purification process using SOURCE Q 16ml and SOURCE ISO 16 ml, as described in EXAMPLE 2. The purity measuredby reverse phase HPLC was 95.8%. (FIG. 5)

Example 7 Preparation of Ser12 mutated DAexendin-4(Lys27)-PEG-immunoglobulin Fc conjugate

3.4K PropionALD(2) PEG and the lysine (Lys) residue of the Ser12 mutatedDA exendin-4 were subjected to pegylation by reacting the peptide andthe 3.4K PropionALD(2) at 25° C. for 3 hours at a molar ratio of 1:30,with a peptide concentration of 3 mg/ml. At this time, the reaction wasperformed in a Na-Phosphaste buffer at pH 7.5 at a concentration of 100mM, and 20 mM SCB as a reducing agent was added thereto to perform thereaction. The purification process of a mono-pegylated peptide usingSOURCE Q (XK 16 ml, Amersham Biosciences) without using SOURCE S (XK 16ml, Amersham Bioscience) was performed as described in EXAMPLE 4. Thereaction was performed at a ratio of peptide:immunoglobulin Fc of 1:8,and a total concentration of proteins of 60 mg/ml at 4° C. for 20 hours.The reaction was performed in a solution of 100 mM K—P (pH 6.0), and 20mM SCB was added as a reducing agent. Because Ser12 mutated DA exendin-4has much stronger anionic property, the excess amounts of immunoglobulinFc, which were not participated in the reaction, were eliminatedeffectively by the purification process using SOURCE Q only.Accordingly, the purification process using SOURCE ISO was omitted, andthe condition of purification process using SOURCE Q was the same as inEXAMPLE 2. The purity measured by reverse phase HPLC was 92.5%. (FIG. 6)

Example 8 Preparation of Arg12 mutated DAexendin-4(Lys27)-PEG-immunoglobulin Fc conjugate

Using the same method as described in EXAMPLE 7, 3.4K PropionALD(2) PEGwas reacted with the lysine (Lys) residue of the Arg12 mutated DAexendin-4(AP, USA), and purified. Then, coupling process was proceeded.The reaction was performed at a ratio of peptide:immunoglobulin Fc of1:8, and a total concentration of proteins of 60 mg/ml at 4° C. for 20hours. The reaction was performed in a solution of 100 mM K—P (pH 6.0),and 20 mM SCB was added as a reducing agent. After the couplingreaction, the two-step purification process using SOURCE Q 16 ml andSOURCE ISO 16 ml was the same as in EXAMPLE 2. The purity measured byreverse phase HPLC was 99.2%. (FIG. 7)

Example 9 Preparation of des amino-histidyl exendin-4(Lys27)-PEG-albuminconjugate

Using the same method as described in EXAMPLE 4, 3.4K PropionALD(2) PEGwas reacted with the lysine (Lys) residue of the des-amino-histidylexendin-4 (AP, USA), and purified. The reaction was performed at a ratioof peptide:human blood-derived albumin (Green cross, South Korea) as acarrier substance of 1:7, and a total concentration of proteins of 50mg/ml at 4° C. for 24 hours. The reaction was performed in a solution of100 mM K—P (pH 6.0), and 20 mM SCB was added as a reducing agent. Afterthe coupling reaction, the two-step purification process using SOURCE Q16 ml and SOURCE ISO 16 ml was the same as in EXAMPLE 2. The puritymeasured by reverse phase HPLC was 90.3%. (FIG. 8)

Example 10 Preparation of dimethyl-histidylexendin-4(Lys27)-PEG-immunoglobulin Fc conjugate

Using dimethyl-histidyl exendin-4 (DM exendin, AP, USA),dimethyl-histidyl exendin-4(Lys27)-immunoglobulin Fc conjugate wasprepared in the same method as described in EXAMPLE 4. The puritymeasured by reverse phase HPLC was 96.4%. (FIG. 9)

Example 11 Preparation of GLP-1 (N)-PEG-immunoglobulin Fc conjugate

3.4K ButyrALD(2) PEG (PEG having two butyraldehyde groups, Nektar, USA)and the N-terminus of the GLP-1 (AP, USA) were subjected to pegylationby reacting the peptide and the PEG at 4° C. for 90 min at a molar ratioof 1:5, with a peptide concentration of 3 mg/ml. At this time, thereaction was performed in a solution of 100 mM K—P (pH 6.0), and 20 mMSCB (NaCNBH₃) as a reducing agent was added thereto. Then, the reactionwas performed at a molar ratio of peptide:immunoglobulin Fc of 1:10, anda total concentration of proteins of 50 mg/ml at 4° C. for 16 hours. Thereaction was performed in a solution of 100 mM K—P (pH 6.0), and 20 mMSCB was added as a reducing agent. After the coupling reaction, thetwo-step purification process using SOURCE Q 16 ml and SOURCE ISO 16 mlwas the same as in EXAMPLE 2. The purity measured by reverse phase HPLCwas 91%. (FIG. 10)

Example 12 Preparation ofdes-amino-histidyl-GLP-1(Lys27)-PEG-immunoglobulin Fc conjugate

3.4K PropionALD(2) PEG and the lysine (Lys) residue of thedes-amino-histidyl GLP-1(AP., USA) were subjected to pegylation byreacting the peptide and the 3.4K PropionALD(2) at 4° C. for 4 hours ata molar ratio of 1:30, with a peptide concentration of 3 mg/ml. At thistime, the reaction was performed in a Na-Phosphaste buffer at pH 7.5 ata concentration of 100 mM, and 20 mM SCB as a reducing agent was addedthereto to perform the reaction. The purification process of amono-pegylated peptide using SOURCE Q (XK 16 ml, Amersham Biosciences)was performed. The reaction was performed at a ratio ofpeptide:immunoglobulin Fc of 1:6, and a total concentration of proteinsof 60 mg/ml at 4° C. for 16 hours. The reaction was performed in asolution of 100 mM K—P (pH 6.0), and 20 mM SCB was added as a reducingagent. After the coupling reaction, the two-step purification processusing SOURCE Q 16 ml and SOURCE ISO 16 ml was the same as in EXAMPLE 2.However, the resolution in SOURCE ISO 16 ml was declined because thedifference of hydrophobicity between GLP-1 immunoglobulin Fc conjugateand immunoglobulin Fc is less than that between exendin-4 immunoglobulinFc and immunoglobulin Fc. Accordingly, the purifying process usingSOURCE ISO 16 ml column was performed further to the above purificationprocess. The purity measured by reverse phase HPLC was 91.9%. (FIG. 11)

Example 13 Preparation of Conjugate Using ButyrALD Linker Peg

Using 3.4K ButyrALD(2) PEG (PEG having two butyraldehyde groups, Nektar,USA), 3.4K-exendin-4 was prepared in the same method as described inEXAMPLE 1. It was coupled immunoglobulin Fc in the same method asdescribed in EXAMPLE 3. The purity measured by reverse phase HPLC was92.3%. (FIG. 12)

Example 14 Measurement of In-Vitro Activity of Sustained ReleaseExendin-4

To measure the efficacy of long acting preparation of exendin-4, amethod for measuring the in-vitro cell activity was used. Typically, inorder to measure the in-vitro activity of GLP-1, insulinoma cells orislet of Langerhans were separated, and whether cAMP's in the cell wasincreased after treatment of GLP-1 was determined.

For the method for measuring the in-vitro activity used in the presenttest, RIN-m5F (ATCC.) cells, which are known as Rat insulinoma cells,were used. These cells have GLP-1 receptors, and thus they are oftenused in the methods for measuring the in-vitro activity in the GLP-1family. RIN-m5F was treated with GLP-1, exendin-4, and test materials atvarying concentrations. The occurrence of cAMP's, which are signalingmolecules in the cells, by the test materials, was measured, and henceEC50 values, and compared to each other. The result was shown in Table1.

TABLE 1 Blood half-life In vitro titer Test Materials (hours) (%)Exendin-4 0.7 100   Exendin-4(N)-PEG-Fc 62 <0.2  Exendin-4(Lys27)-PEG-Fc 61 13.2  DM exendin-4(Lys27)-PEG-Fc 69 2.6 DAexendin-4(Lys27)-PEG-Fc 54 13.2  HY exendin-4(Lys27)-PEG-Fc 52 7.6 CAexendin-4(Lys27)-PEG-Fc 52 8.5 Ser12 DA exendin-4(Lys27)-PEG-Fc N.D. 2.6DA exendin-4(Lys27)-PEG-albumin 2.0 DA GLP-1(Lys20, 28)-PEG-Fc 27 2.0

-   -   DM exendin-4: Dimethyl-histidyl exendin-4    -   DA exendin-4: Des-amino-histidyl exendin-4    -   HY exendin-4: Beta-hydroxy-imidazo-propionyl exendin-4    -   CA exendin-4: imidazo-acetyl exendin-4    -   Ser12 DA exendin-4: DA exendin-4 wherein the 12th lysine residue        of the exendin-4 is substituted with Serine    -   DA GLP-1: des-amino-histidyl-GLP-1    -   Exendin-4(N)-PEG-Fc: Conjugate in which the N-terminus of the        exendin-4 and the Fc region were linked to PEG    -   Exendin-4(Lys27)-PEG-Fc: Conjugate in which the 27th lysine        residue of the exendin-4 and the Fc region were linked to PEG.    -   DM exendin-4(Lys27)-PEG-Fc: Conjugate in which the 27th lysine        residue of the dimethyl histidyl exendin-4 and the Fc region        were linked to PEG.    -   DA exendin-4(Lys27)-PEG-Fc: Conjugate in which the 27th lysine        residue of the des-amino-histidyl exendin-4 and the Fc region        were linked to PEG.    -   HY exendin-4(Lys)-PEG-Fc: Conjugate in which the 27th lysine        residue of the beta-hydroxy-imidazo-propionyl exendin-4 and the        Fc region were linked to PEG.    -   CA exendin-4(Lys)-PEG-Fc: Conjugate in which the 27th lysine        residue of the imidazo-acetyl exendin-4 and the Fc region were        linked to PEG.    -   Ser12 DA exendin-4(Lys27)-PEG-Fc: Conjugate in which the 27th        lysine residue of the Ser12 des-amino-histidyl exendin-4 wherein        the 12th lysine residue of the exendin-4 is substituted for        Serine and the Fc region were linked to PEG.    -   DA exendin-4(Lys27)-PEG-Albumin: Conjugate in which the 27th        lysine residue of the des-amino-histidyl exendin-4 and albumin        were linked to PEG.    -   DA GLP-1(Lys20, 28)-PEG-Fc: Conjugate in which the lysine        residue of the des-amino-histidyl GLP-1 and the Fc region were        linked to PEG.

Example 15 Test of In-Vivo Efficacy of the Long Acting Exendin-4

To measure the in-vivo efficacy of long acting preparation theexendin-4, a method for measuring the effect of reducing the glucoseconcentration in blood for db/db mice as diabetic models was used. 100mcg/kg of long acting exendin-4 preparation was administered once duringa 2 week period, and 100 mcg/kg of native exendin-4 was administered perday, to about 6-7-week-old diabetic model mice, without limiting thefood supply. After administering the test materials, blood was collecteddaily, and the change of blood glucose level was measured. Especially,in case of the native exendin-4, the blood glucose concentration wasmeasured after 1 hour of the administration (FIG. 14). The conjugates ofthe exendin-4 derivatives maintained reduction of the blood glucoseconcentration for 10 days or longer even when administered once, whilethe glucose lowering activity of the conjugates of the native exendin-4disappeared after 8 days.

INDUSTRIAL APPLICABILITY

The insulinotropic peptide conjugate of the present invention has thein-vivo activity which is maintained relatively high, and has remarkablyincreased blood half-life, and thus it can be desirably employed in thedevelopment of long acting formulations of various peptide drugs.

1. An insulinotropic peptide conjugate, comprising an insulinotropicpeptide, a non-peptidyl polymer, and an immunoglobulin Fc fragment oralbumin, wherein the insulinotropic peptide, and the immunoglobulin Fcfragment or albumin are covalently linked through the non-peptidylpolymer, wherein the non-peptidyl polymer is selected from the groupconsisting of polyethylene glycol, polypropylene glycol, copolymers ofethylene glycol and propylene glycol, polyoxyethylated polyols,polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether,biodegradable polymers, lipid polymers, chitins, hyaluronic acid, andcombinations thereof, and wherein one end of the non-peptidyl polymer islinked to an amino acid residue other than the N-terminus of theinsulinotropic peptide.
 2. The insulinotropic peptide conjugateaccording to claim 1, wherein the insulinotropic peptide is selectedfrom the group consisting of GLP-1, an exendin-3, an exendin-4, anagonist, derivative, fragment, and variant thereof, and combinationsthereof.
 3. The insulinotropic peptide conjugate according to claim 2,wherein the derivative is a peptide having at least 80% amino acidsequence homology with the native insulinotropic peptide and possessingan insulinotropic function and having an amine group at the N-terminusof the native insulinotropic peptide substituted, deleted, or modified,and a fragment and variant thereof.
 4. The insulinotropic peptideconjugate according to claim 2, wherein the derivative is selected fromthe group consisting of a peptide possessing deleted alpha carbon and anamine group at the N-terminus of the native insulinotropic peptide, anda fragment and variant thereof.
 5. An insulinotropic peptide conjugateaccording to claim 2, wherein the derivative of exendin-4 derivativehaving at least 80% amino acid sequence homology with the nativeexendin-4 and possessing an insulinotropic function, and wherein thederivative is an exendin-4 derivative selected from the group consistingof a peptide having an amine group at the N-terminus of the nativeexendin-4 substituted, deleted or modified, a peptide having deletedalpha carbon and an amine group at the N-terminus of the nativeexendin-4, and a fragment and variant thereof.
 6. The insulinotropicpeptide conjugate according to claim 5, wherein the exendin-4 derivativeis selected from the group consisting of an exendin-4 derivativeprepared by deleting N-terminal amine group, an exendin-4 derivativeprepared by substituting N-terminal amine group with hydroxyl group, anexendin-4 derivative prepared by modifying N-terminal amine group withdimethyl group, an exendin-4 derivative prepared by deleting α-carbon ofhistidine, the first amino acid of exendin-4 among the insulinotropicpeptides.
 7. An insulinotropic peptide conjugate according to claim 2,wherein the variant is an exendin-4 variant prepared by substituting12th amino acid (lysine) for serine, or an exendin-4 variant prepared bysubstituting 12th amino acid (lysine) with arginine. 8-12. (canceled)13. An insulinotropic peptide derivative, wherein the insulinotropicderivative is represented by the following Chemical Formula 1: Aninsulinotropic peptide derivative, wherein the insulinotropic derivativeis represented by the following Chemical Formula 1:R₁—X—Y—Z—R₂  <Formula 1> Wherein, R₁ is selected from the groupconsisting of histidine, des-amino-histidyl group, N-dimethyl-histidylgroup, beta-hydroxy imidazopropyl group and 4-imidazoacetyl group; R₂ isselected from the group consisting of —NH₂, —OH and -Lys; X is selectedfrom the group consisting ofGly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-R3-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-11e-Glu-Trp-Leu-R4-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser,Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-R3-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-R4-Asn-Gly-Gly,andSer-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-R3-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-R4-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser;R₃ is selected from the group consisting of Lys, Ser and Arg; R₄ isselected from the group consisting of Lys, Ser and Arg; Y ispolyethylene glycol, polypropylene glycol, copolymers of ethylene glycoland propylene glycol, polyoxyethylated polyols, polyvinyl alcohol,polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers,lipid polymers, chitins, hyaluronic acid, and combinations thereof; andZ is an immunoglobulin Fc region or albumin.
 14. The insulinotropicpeptide conjugate according to claim 1, wherein the non-peptidyl polymerhas both ends, each binding to an amine group or a thiol group of theimmunoglobulin Fc region, and the insulinotropic peptide.
 15. Theinsulinotropic peptide conjugate according to claim 1, wherein theimmunoglobulin Fc region is deglycosylated.
 16. The insulinotropicpeptide conjugate according to claim 1, wherein the immunoglobulin Fcregion is composed of one to four domains selected from the groupconsisting of C_(H)1, C_(H)2, C_(H)3 and C_(H)4 domains.
 17. Theinsulinotropic peptide conjugate according to claim 16, wherein theimmunoglobulin Fc region further includes a hinge region.
 18. Theinsulinotropic peptide conjugate according to claim 1, wherein theimmunoglobulin Fc region is an Fc region derived from an immunoglobulinselected from the group consisting of IgG, IgA, IgD, IgE, and IgM. 19.The insulinotropic peptide conjugate according to claim 18, wherein eachdomain of the immunoglobulin Fc region is a domain hybrid of a differentorigin derived from an immunoglobulin selected from the group consistingof IgG, IgA, IgD, IgE, and IgM.
 20. The insulinotropic peptide conjugateaccording to claim 18, wherein the immunoglobulin Fc region is a dimeror a multimer (a combination of immunoglobulin Fc) composed ofsingle-chain immunoglobulins of the same origin.
 21. The insulinotropicpeptide conjugate according to claim 18, wherein the immunoglobulin Fcregion is an IgG4 Fc region.
 22. The insulinotropic peptide conjugateaccording to claim 21, wherein the immunoglobulin Fc region is a humandeglycosylated IgG4 Fc region.
 23. The insulinotropic peptide conjugateaccording to claim 1, wherein the reactive group of the non-peptidylpolymer is selected from the group consisting of an aldehyde group, apropionaldehyde group, a butyraldehyde group, a maleimide group, and asuccinimide derivative.
 24. The insulinotropic peptide conjugateaccording to claim 23, wherein the succinimide derivative is selectedfrom the group consisting of succinimidyl propionate, succinimidylcarboxymethyl, hydroxy succinimidyl, or succinimidyl carbonate.
 25. Theinsulinotropic peptide conjugate according to claim 23, wherein thenon-peptidyl polymer has a reactive aldehyde group at both ends.
 26. Theinsulinotropic peptide conjugate according to claim 25, wherein thenon-peptidyl polymer is polyethylene glycol.
 27. A method for preparingan insulinotropic peptide conjugate, comprising the steps of: (1)covalently linking a non-peptidyl polymer having a reactive groupselected from the group consisting of aldehyde, maleimide, andsuccinimide derivatives at both ends thereof, with an amine or thiolgroup of an insulinotropic peptide; (2) isolating a conjugate comprisingthe insulinotropic peptide from the reaction mixture of (1), in whichthe non-peptidyl polymer is linked covalently to an amino acid otherthan the amino acid at the N-terminus; and (3) covalently linking animmunoglobulin Fc region or albumin to the other end of the non-peptidylpolymer of the isolated conjugate to produce a peptide conjugatecomprising the immunoglobulin Fc region and the insulinotropic peptide,which are linked to each end of the non-peptide polymer.
 28. A methodfor preparing an insulinotropic peptide conjugate, comprising the stepsof: (1) covalently linking a non-peptidyl polymer having an aldehydereactive group at both ends thereof with the lysine residue of theinsulinotropic peptide at pH of 7.5 or more; (2) isolating a conjugatecomprising the insulinotropic peptide from the reaction mixture of (1),in which the non-peptidyl polymer is linked covalently to the lysineresidue; and (3) covalently linking an immunoglobulin Fc region to theother end of the non-peptidyl polymer of the isolated conjugate toproduce a protein conjugate comprising the immunoglobulin Fc region andthe insulinotropic peptide, which are linked to each end of thenon-peptidyl polymer.
 29. The method for preparing an insulinotropicpeptide conjugate according to claim 28, wherein the insulinotropicpeptide is selected from the group consisting of a exendin-4 derivateprepared by deleting an amine group at the N terminus, an exendin-4derivative prepared by substituting N-terminal amine group with hydroxylgroup, an exendin-4 derivative prepared by modifying N-terminal aminegroup with dimethyl group, an exendin-4 derivative prepared by deletingα-carbon of histidine, the first amino acid of exendin-4, an exendin-4variant prepared by substituting 12th amino acid (lysine) for serine,and an exendin-4 variant prepared by substituting 12th amino acid(lysine) with arginine.
 30. The method for preparing an insulinotropicpeptide conjugate according to claim 28, wherein the insulinotropicpeptide is selected from the group consisting of a exendin-4 derivateprepared by deleting an amine group at the N terminus, an exendin-4derivative prepared by substituting N-terminal amine group with hydroxylgroup, an exendin-4 derivative prepared by modifying N-terminal aminegroup with dimethyl group, an exendin-4 derivative prepared by deletingα-carbon of histidine, the first amino acid of exendin-4, an exendin-4variant prepared by substituting 12th amino acid (lysine) for serine,and an exendin-4 variant prepared by substituting 12th amino acid(lysine) with arginine. 31-32. (canceled)
 33. A pharmaceuticalcomposition for treating diabetes, obesity, acute coronary syndrome, orpolycystic ovary syndrome, comprising the peptide conjugate of claim 1.